Permeability of lipophilic compounds in drug discovery using in-vitro human absorption model, Caco-2.

Highly lipophilic compounds are often encountered in the early stages of drug discovery. The apparent permeability (Papp) of these compounds in Caco-2 cell could be underestimated because of considerable retention by the Caco-2 monolayer and non-specific binding to transwell surface. We have utilized a general approach for the determination of permeability of these compounds, which includes the addition of 1-5% DMSO in the apical (AP) and 4% bovine serum albumin (BSA) in the basolateral (BA) side. Two highly lipophilic and highly protein bound Schering compounds, SCH-A and SCH-B, exhibited poor recovery and low Papp in the conventional Caco-2 system that included 1% DMSO in the AP and BA sides. In contrast, both compounds were well absorbed in cynomolgus monkeys. Inclusion of BSA (up to 4%) in the BA side provided necessary absorptive driving force similar to in vivo sink conditions improving both recovery and Papp of these compounds as well as progesterone, a model highly lipophilic and highly protein bound compound. Whereas, the recovery and Papp of mannitol (high recovery, low permeability) and propranolol (high recovery, high permeability) remained unaffected. The presence of 4% BSA increased Papp of SCH-A, SCH-B, and progesterone by five-, four-, and three-fold, respectively. We also compared this approach with a second, based on the disappearance of the compound from the AP side, which resulted in a reasonable estimate of the permeability (23.3x10(-6) cm/s) for SCH-A. The results demonstrated that the reliable estimates of permeability of highly lipophilic compounds that are subjected to considerable retention by the cell monolayer and exhibit non-specific binding are obtained by the addition of BSA to the BA side.

Mechanism-based inactivation of CYP2D6 by 5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine.

SCH 66712 [5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine] caused a time- and NADPH-dependent loss of CYP2D6 activity. The inactivation of human liver (HL) microsomal dextromethorphan O-demethylase activity, a prototype marker for CYP2D6, was characterized by a K(I) of 4.8 microM and a maximal rate constant of inactivation (k(inact)) of 0.14 min(-1). The inactivation of the recombinant CYP2D6 in Supersomes (r-CYP2D6) was characterized by a K(I) of 0.55 microM and a k(inact) of 0.32 min(-1). Extensive dialysis of the SCH 66712-inhibited enzyme failed to restore the activity to control levels (dialyzed reaction mixture lacking SCH 66712) for both HL microsomes and r-CYP2D6. Addition of glutathione, superoxide dismutase, or mannitol to the reaction mixture failed to protect CYP2D6 against SCH 66712-NADPH-catalyzed inactivation. Addition of quinidine, a reversible inhibitor of CYP2D6, to a preincubation mixture consisting of SCH 66712, HL microsomes, or Supersomes and NADPH partially protected CYP2D6 from inactivation. SCH 66712 also inhibited HL microsomal CYP3A4, CYP2C9, and CYP2C19; however, the concentrations required to inhibit those isoforms were 5- to 10-fold higher than those required to inhibit CYP2D6. These results demonstrate that SCH 66712 is a potent and fairly selective mechanism-based inhibitor of CYP2D6.

Inhibition of CYP3A4 in a rapid microtiter plate assay using recombinant enzyme and in human liver microsomes using conventional substrates.

Cytochrome P450 inhibition studies are performed in the pharmaceutical industry in the discovery stage to screen candidates that may have the potential for clinical drug-drug interactions. A 96-well microtiter plate assay using recombinant cytochrome P450 (Supersomes) has been used to increase the overall throughput. The IC(50) values for the inhibition of CYP3A4 by 52 new chemical entities (NCEs) were determined using the Supersomes assay with resorufin benzyl ether as a substrate, and the data were compared with those obtained in human liver microsomes (HLM) using midazolam as a substrate. Among the 52 compounds tested, 25 showed IC(50) values within a 5-fold difference in the two assays. For all compounds that showed a >5-fold difference, the IC(50) values in the Supersomes assay were lower than those obtained in HLM, except for one compound. Further studies suggested that this discrepancy was not related to difference in protein concentrations between the two assays. In addition, the IC(50) values for 16 compounds with a wide range of inhibition potency were determined in HLM using testosterone and dextromethorphan as substrates. The results showed an 80 to 93% match within a 5-fold difference between the three probe substrates. However, for certain compounds including ketoconazole, there were substrate-dependent differences in the inhibition. The results suggest that the difference between the Supersomes and HLM could be partially attributed to differences in the substrate used, and to metabolism by other cytochrome P450s present in the HLM but not in the Supersomes. Furthermore, multiple CYP3A4 substrates should be used to improve the reliability of estimating potential drug-drug interaction of NCEs.

High-performance liquid chromatographic analysis and stability of anti-tumor agent temozolomide in human plasma.

Temozolomide (SCH 52365; TEMODAL) is an antineoplastic agent with activity against a broad spectrum of murine tumors. This compound is currently marketed in the European Union for the treatment of patients with glioblastoma multiforme and anaplastic astrocytoma, which are serious and aggressive types of brain cancers. It has been postulated that temozolomide exerts its in vivo activity via the decomposition product MTIC, which is believed to alkylate nucleophiles, and in the process is converted to AIC. A high-performance liquid chromatographic (HPLC) method was developed and validated for the analysis of temozolomide in human plasma. The determination of temozolomide involved extraction with ethyl acetate followed by separation on a reversed phase C-18 column and quantification by UV absorbance at 316 nm. The calibration curve was linear over a concentration range of 0.1-20 microg/ml. The limit of quantitation was 0.1 microg/ml, where the coefficient of variation (CV) was 0% and the bias was 10.0%. The method was precise with a coefficient of variation ranging from 2.5 to 6.9% and accurate with a bias ranging from 5.0 to 10.0%. Temozolomide was unstable at 37 degrees C in human plasma with a degradation t1/2 of 15 min; however, it was stable at 4 degrees C for at least 30 min. Temozolomide was stable in acidified human plasma (pH < 4) for at least 24 h at 25 degrees C, and for at least 30 days at -20 degrees C. Moreover, temozolomide was stable in acidified human plasma after being subjected to three freeze thaw cycles. The assay was shown to be specific, accurate, precise, and reliable for use in pharmacokinetic studies.

Chiral high-performance liquid chromatographic analysis of antifungal SCH 56592 and evaluation of its chiral inversion in animals and humans.

SCH 56592 is a novel triazole antifungal agent that is active both orally and intravenously in animal models of infection. This compound is in Phase II-III clinical trials for the treatment of systemic fungal infections. SCH 56592 is a single enantiomer with four stereogenic centers; therefore, it was necessary to evaluate the possible chiral inversion of this drug candidate in animals and humans. Thus, chiral high-performance liquid chromatographic (HPLC) methods have been developed to separate SCH 56592 from its diastereomers and to evaluate its chiral inversion in rats, dogs, cynomolgus monkeys, and humans. Chiral HPLC analysis involved the use of a Chiralcel OD column set at 39 degrees C with a mobile phase of hexane-ethanol-diethylamine and a fluorescence detector set at an excitation wavelength of 270 nm and an emission wavelength of 390 nm. Plasma or serum samples were subjected to solid phase extraction on a C(2) cartridge followed by HPLC analysis. The method was sensitive with a limit of quantitation of 0.1 microg/ml in dog serum. The linearity was satisfactory, as shown by correlations of >0.997 and by visual examination of the calibration curves. The precision and accuracy were satisfactory, as indicated by coefficients of variation (CV) ranging from 1.1 to 12.1% and bias values ranging from -11.0 to 9.0%. Chiral HPLC analysis indicated that SCH 56592 was not subjected to chiral inversion in rats, dogs, cynomolgus monkeys, and humans.

High-performance liquid chromatographic analysis of the anti-fungal agent SCH 56592 in dog serum.

SCH 56592 is a novel triazole antifungal agent that is active both orally and intravenously. This compound is in phase II-III clinical trials for the treatment of systemic fungal infections. A high-performance liquid chromatographic (HPLC) method was developed for the analysis of SCH 56592 in serum of dogs, a species used for safety evaluation. The HPLC analysis involved protein precipitation with methanol followed by separation on a C18 column and quantitation by UV absorbance at 262 nm. The method was sensitive with a limit of quantification of 0.05 microg/ml in dog serum. The linearity was satisfactory as indicated by correlations of >0.996, in addition to visual examination of the calibration curves. The precision and accuracy were satisfactory as indicated by coefficients of variation (C.V.) ranging from 2.0 to 3.8%, and bias values ranging from -6.5 to 10%. Moreover, SCH 56592 was stable in dog serum after being subjected to three freeze-thaw cycles. The assay was shown to be sensitive, specific, accurate, precise, and reliable for use in pharmacokinetic or toxicokinetic studies.

Pharmacokinetics of SCH 56592, a new azole broad-spectrum antifungal agent, in mice, rats, rabbits, dogs, and cynomolgus monkeys.

SCH 56592 is a new broad-spectrum azole antifungal agent that is in phase 3 clinical trials for the treatment of serious systemic fungal infections. The pharmacokinetics of this drug candidate were evaluated following its intravenous (i.v.) or oral (p.o.) administration as a solution in hydroxypropyl-beta-cyclodextrin (HPbetaCD) or oral administration as a suspension in 0.4% methylcellulose (MC) in studies involving mice, rats, rabbits, dogs, and cynomolgus monkeys. SCH 56592 was orally bioavailable in all species. The oral bioavailability was higher with the HPbetaCD solution (range, 52 to approximately 100%) than from the MC suspension (range, 14 to 48%) and was higher in mice ( approximately 100% [HPbetaCD] and 47% [MC]), rats ( approximately 66% [HPbetaCD] and 48% [MC]), and dogs (72% [HPbetaCD] and 37% [MC]) than in monkeys (52% [HPbetaCD] and 14% [MC]). In rabbits, high concentrations in serum suggested good oral bioavailability with the MC suspension. The i.v. terminal-phase half-lives were 7 h in mice and rats, 15 h in dogs, and 23 h in monkeys. In rabbits, the oral half-life was 9 h. In species given increasing oral doses (mice, rats, and dogs), serum drug concentrations were dose related. Food produced a fourfold increase in serum drug concentrations in dogs. Multiple daily doses of 40 mg of SCH 56592/kg of body weight for eight consecutive days to fed dogs resulted in higher concentrations in serum, indicating accumulation upon multiple dosing, with an accumulation index of approximately 2.6. Concentrations above the MICs and minimum fungicidal concentrations for most organisms were observed at 24 h following a single oral dose in MC suspension in all five species studied (20 mg/kg for mice, rats, and rabbits and 10 mg/kg for dogs and monkeys), suggesting that once-daily administration of SCH 56592 in human subjects would be a therapeutically effective dosage regimen.

Validation of higher-throughput high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry assays to conduct cytochrome P450s CYP2D6 and CYP3A4 enzyme inhibition studies in human liver microsomes.

In the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single drug candidate can be identified for development. In order to accelerate the drug discovery process, we have developed higher-throughput enzyme assays to evaluate the inhibition of cytochrome P450 isoforms 2D6 (CYP2D6) and 3A4 (CYP3A4) in human liver microsomes. The assays are based on high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) techniques. The analysis time for each sample was reduced from approximately 20 minutes for the conventional HPLC assay to 30 seconds for the LC/MS/MS assay. For both LC/MS/MS assays, the linearity (r(2) > 0.99), precision (%CV < 15%) and accuracy (% bias <15%) for both inter- and intraday validations were satisfactory. Since the implementation of the LC/MS/MS assays, our sample throughput has increased by over 40-fold.

Stimulation of tolbutamide hydroxylation by acetone and acetonitrile in human liver microsomes and in a cytochrome P-450 2C9-reconstituted system.

Organic solvents are often used to solubilize lipophilic new chemical entities before their addition to in vitro test systems such as microsomal stability or cytochrome P-450 (CYP) inhibition. However, the effect of these organic solvents on the test systems is not usually characterized. This study was initiated to evaluate the effect of acetonitrile and acetone, in addition to other organic solvents, on the tolbutamide hydroxylation activity of CYP2C9 in both human liver microsomes and a CYP2C9-reconstituted system. Both acetonitrile and acetone significantly stimulated the NADPH-dependent tolbutamide hydroxylation by nearly 2- to 3-fold in human liver microsomes and CYP2C9-reconstituted system when incubated at 2 and 4% final solvent concentrations. When cumene hydroperoxide was used instead of NADPH, both acetone and acetonitrile significantly inhibited tolbutamide hydroxylation. This NADPH-dependent stimulatory effect was further evaluated by examining the effect of a series of other organic solvents with different carbon chain lengths and various functional groups, including hydroxyl, ketone, and aldehyde. Unlike acetone, two other ketone-containing solvents, methyl ethyl ketone (2-butanone) and diethyl ketone (3-pentanone) failed to significantly enhance tolbutamide hydroxylation. Other solvents tested, including methanol, ethanol, propanol, 1-butanol, 2-butanol, 1-pentanol, 2-pentanol, acetaldehyde, and dimethyl sulfoxide significantly inhibited NADPH-dependent tolbutamide hydroxylation. Overall, the stimulatory effect of both acetonitrile and acetone on tolbutamide hydroxylation was found to be primarily due to a consistent increase in V(max), whereas K(m) was unchanged in both human liver microsomes and the reconstituted CYP2C9 system. These data suggest that acetone and acetonitrile stimulate NADPH-mediated tolbutamide hydroxylation via the CYP reductase and not by modifying the affinity of tolbutamide for the CYP2C9 enzyme.

Analogues of 1-(3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo-[5,6]-cyclohepta [1,2-b]pyridin-11-yl)piperidine as inhibitors of farnesyl protein transferase.

The synthesis of several 4-pyridylacetyl N-oxide derivatives of 4-(3-bromo-6,11-dihydro-5H-benzo[5,6]-cyclohepta[1,2-b]-pyridin-11-yl)pi perazine/piperidine 3 is described. This study was aimed at identifying fomesyl protein transferase (FPT) inhibitors in these two series of tricycles containing different phenyl ring substituents. The in vitro activity profile of the initial group of compounds 7a-7g led to the synthesis of the 8-methyl-10-methoxy and 8-methyl-10-bromo analogues 7i, 13i, and 13j. The 11R(-) enantiomers of these compounds were found to exhibit potent in vitro FPT inhibition activity.

Atropisomeric trihalobenzocycloheptapyridine analogues provide stereoselective FPT inhibitors with antitumor activity.

Introduction of bromine at the 10-position of 3-bromo-8-chloro-benzocycloheptapyridine analogues of type 3 results in formation of atropisomeric compounds of type (+/-)-1 and (+/-)-2 that are easily separable at room temperature on a ChiralPak AD column providing pure atropisomers, (+)-1, (-)-1, and (+)-2, (-)-2, respectively. Evaluation of the FPT activity of these atropisomers revealed that compounds (+)-1 and (+)-2 were more potent in the FPT enzyme and cellular assay than their (-)-isomer counterparts. Compounds (+)-1 and (+)-2 were found to inhibit FPT processing in COS cells at low micro molar range. They were also found to have excellent cellular antitumor activity. Evaluation of compound (+)-1 and (+)-2 in DLD-tumor model in nude mice revealed that they were efficacious, inhibiting tumor growth by 55 and 63% at 50 mpk, respectively.

High-performance liquid chromatographic analysis of the anti-tumor agent SCH 66336 in cynomolgus monkey plasma and evaluation of its chiral inversion in animals.

SCH 66336 is a novel non-cytotoxic anti-tumor agent that is in phase I/II clinical trials for the treatment of solid tumors. This compound is a single enantiomer with one chiral center. Prior to evaluation of this drug candidate in man, it was necessary to evaluate its pharmacokinetics and possible chiral inversion in animals. Thus, high-performance liquid chromatographic (HPLC) methods have been developed for its determination in cynomolgus monkey plasma and for the evaluation of its chiral inversion in rats and cynomolgus monkeys. The achiral HPLC analysis involved extraction with 30% methylene chloride in hexane followed by separation on a CN column and quantitation by UV absorbance at 280 nm. The method was linear over a concentration range of 0.1 to 20 microg/ml in monkey plasma. The chiral HPLC analysis involved the use of a Chiralpak AD column set at 39 degrees C with a mobile phase of hexane-ethanol-diethylamine mixture and a UV detector set at 280 nm. Plasma samples were subjected to solid-phase extraction on a C2 cartridge prior to HPLC analysis. The method was linear over a concentration range of 0.25 to 10 microg/ml in rat and cynomolgus monkey plasma for both enantiomers. Both methods showed good linearity (r2>0.99), accuracy (bias< 13%) and precision (CV<12%). Chiral HPLC analysis indicated that SCH 66336 was not subjected to chiral inversion in rats and cynomolgus monkeys.

Improved reliability of the rapid microtiter plate assay using recombinant enzyme in predicting CYP2D6 inhibition in human liver microsomes.

A higher throughput method of screening for the inhibition of recombinant CYP2D6 using a microtiter plate (MTP) assay was evaluated using 62 new chemical entities and compared to data from the dextromethorphan O-demethylase assay in human liver microsomes (HLM). The IC50 values for the two assays closely matched for 53 compounds (85%). Six of the variant nine compounds had higher IC50 values with the recombinant enzyme, whereas three had lower IC50 values with the recombinant enzyme. When the inhibition with the recombinant enzyme was determined at various time points, the IC50 values increased as the duration of the incubation increased for the six compounds with higher IC50 values in the MTP assay. The IC50 values at 10 min matched more closely the IC50 values in HLM (95% compared with 85%). For three compounds that showed comparable IC50 values in the two assays, and the three compounds with lower IC50 values in the MTP assay, the IC50 values did not change over time. These results suggest that the six compounds that showed higher IC50 values in the MTP assay at 45 min are substrates for CYP2D6. Using known CYP2D6 substrates, a similar phenomenon was observed, i.e., inhibition curves shifted to higher IC50 values as incubation time increased. These results indicate that the higher throughput MTP assay is more comparable to HLM if the IC50 values are determined at 10 min rather than the recommended 45 min. Furthermore, data acquisition at multiple time points may indicate if a compound is a potential substrate or metabolism/mechanism-based inhibitor for the enzyme.

Effects of SCH 59228, an orally bioavailable farnesyl protein transferase inhibitor, on the growth of oncogene-transformed fibroblasts and a human colon carcinoma xenograft in nude mice.

The products of the Ha-, Ki-, and N-ras proto-oncogenes comprise a family of 21 kDa guanine nucleotide-binding proteins which play a crucial role in growth factor signal transduction and in the control of cellular proliferation and differentiation. Activating mutations in the ras oncogenes occur in a wide variety of human tumors. Ras proteins undergo a series of posttranslational processing events. The first modification is addition of the 15-carbon isoprene, farnesyl, to a Cys residue near the carboxy-terminus of Ras. Prenylation allows the Ras oncoprotein to localize to the plasma membrane where it can initiate downstream signalling events leading to cellular transformation. Inhibitors of the enzyme which catalyzes this step, farnesyl protein transferase (FPT), are a potential class of novel anticancer drugs which interfere with Ras function. SCH 59228 is a tricyclic FPT inhibitor which inhibits the farnesylation of purified Ha-Ras with an IC50 of 95 nM and blocks the processing of Ha-Ras in Cos cells with an IC50 of 0.6 microM. SCH 59228 has favorable pharmacokinetic properties upon oral dosing in nude mice. The in vivo efficacy of SCH 59228 was evaluated using a panel of tumor models grown in nude mice. These included several rodent fibroblast lines expressing mutationally-activated (val12) forms of the Ha-Ras oncogene. In some cases, these proteins contain their native C-terminal sequence (CVLS) which directs farnesylation. In one model, the C-terminal sequence was altered to CVLL, making the expressed protein a substrate for a distinct prenyl transferase, geranylgeranyl protein transferase-1. When dosed orally at 10 and 50 mg/kg (four times a day, 7 days a week) SCH 59228 significantly inhibited tumor growth of cells expressing farnesylated Ha-Ras in a dose-dependent manner; over 90% growth inhibition was observed at the 50 mg/kg dose. Tumor growth of cells expressing the geranylgeranylated form of Ha-Ras was less potently inhibited. Growth of tumors derived from a rodent fibroblast line expressing activated Ki-Ras containing its native C-terminal sequence (CVIM), which preferentially directs farnesylation, was also inhibited by SCH 59228. Inhibition in the Ki-Ras model was less than that observed in the Ha-Ras model. In contrast, tumors derived from cells transformed with the mos oncogene were not significantly inhibited even at the highest dose level. SCH 59228 also significantly and dose-dependently inhibited the growth of human colon adenocarcinoma DLD-1 xenografts (which express activated Ki-ras). These results indicate that SCH 59228 possesses in vivo antitumor activity upon oral dosing in tumor models expressing activated ras oncogenes. This is the first report of oral antitumor activity with an FPT inhibitor. These results are discussed in light of recent observations on alternative prenylation of some Ras isoforms.

High-performance liquid chromatographic analysis of the D4 receptor antagonist SCH 66712 in rat plasma.

SCH 66712 is a potent and selective dopamine D4 receptor antagonist. An HPLC method was developed for the analysis of SCH 66712 in the plasma of rats, a species used for safety evaluation of this compound. The method involved solid-phase extraction on an ethyl cartridge and HPLC separation on a reversed-phase C8 column with quantitation using a fluorescence detector. The calibration curve was linear over a concentration range of 5-100 ng/ml. The limit of quantitation was 5 ng/ml, where the coefficient of variation (C.V.) was 2.9% and the bias was 6%. The precision of the method was satisfactory as indicated by an intra-day C.V. of < or = 4% and an inter-day C.V. of < or = 6%. The accuracy was also satisfactory as shown by an intra-day bias of < or = 8% and an inter-day bias of < or = 9%. The assay was shown to be sensitive, specific, accurate, precise, and reliable for use in pharmacokinetic or toxicokinetic studies.

Antitumor activity of SCH 66336, an orally bioavailable tricyclic inhibitor of farnesyl protein transferase, in human tumor xenograft models and wap-ras transgenic mice.

We have been developing a series of nonpeptidic, small molecule farnesyl protein transferase inhibitors that share a common tricyclic nucleus and compete with peptide/protein substrates for binding to farnesyl protein transferase. Here, we report on pharmacological and in vivo studies with SCH 66336, a lead compound in this structural class. SCH 66336 potently inhibits Ha-Ras processing in whole cells and blocks the transformed growth properties of fibroblasts and human tumor cell lines expressing activated Ki-Ras proteins. The anchorage-independent growth of many human tumor lines that lack an activated ras oncogene is also blocked by treatment with SCH 66336. In mouse, rat, and monkey systems, SCH 66336 has excellent oral bioavailability and pharmacokinetic properties. In the nude mouse, SCH 66336 demonstrated potent oral activity in a wide array of human tumor xenograft models including tumors of colon, lung, pancreas, prostate, and urinary bladder origin. Enhanced in vivo efficacy was observed when SCH 66336 was combined with various cytotoxic agents (cyclophosphamide, 5-fluorouracil, and vincristine). In a Ha-Ras transgenic mouse model, prophylactic treatment with SCH 66336 delayed tumor onset, reduced the average number of tumors/mouse, and reduced the average tumor weight/animal. In a therapeutic mode in which gavage treatment was initiated after the transgenic mice had developed palpable tumors, significant tumor regression was induced by SCH 66336 in a dose-dependent fashion. This was associated with increased apoptosis and decreased DNA synthesis in tumors of animals treated with SCH 66336. Enhanced efficacy was also observed in this model when SCH 66336 was combined with cyclophosphamide. SCH 66336 is presently being evaluated in Phase I clinical trials.

Liquid chromatographic analysis in mouse, dog and human plasma; stability, absorption, metabolism and pharmacokinetics of the anti-HIV agent 2-chloro-5-(2-methyl-5,6-dihydro-1,4-oxathiin-3-yl carboxamido) isopropylbenzoate (NSC 615985, UC84).

NSC 615985 (UC 84) has demonstrated anti-HIV activity in the NCI-AIDS antiviral screen and was under consideration as an anti-AIDS drug. The compound was subsequently shown to be a non-nucleoside reverse transcriptase inhibitor (NNRTI). An HPLC method was developed for the analysis of NSC 615985 in mouse, dog and human plasma; and was used to study its stability in plasma and blood as well as its absorption and metabolism in mice. The method involved precipitation of plasma protein with three volumes of methanol followed by HPLC analysis of the supernatant. The HPLC analysis was carried out on a reversed-phase Nova-Pak C18 column with a mobile phase of KH2PO4 (0.01 M; pH 4.8)-acetonitrile (52:48, v/v) at a flow rate of 1 ml min-1 and quantification with a UV detector set at 259 nm. The lower limit of quantitation was 0.05 microgram ml-1 in 1 ml of dog or human plasma or 0.1 microgram ml-1 in 0.5 ml of mouse plasma. NSC 615985 was more stable in dog and human plasma than in mouse plasma, and was less stable in blood than in plasma of the three species investigated. Following bolus intravenous (i.v.) administration at 10 mg kg-1 to male CDF1 mice, NSC 615985 elimination followed biexponential kinetics with half-lives of 1 and 7 min, and was extensively metabolized. NSC 615985 was very poorly absorbed following oral (PO) administration as a suspension in water or in 20% lipid emulsion (Liposyn II). Following bolus subcutaneous (s.c.) administration of [14C]NSC 615985 at 10 mg kg-1, relatively low concentrations of the parent compound were observed in three of 36 mice. One metabolite was tentatively identified in plasma of both the i.v.- and s.c.-treated animals as the sulfoxide of the parent compound. No parent compound was detected in the urine of NSC 615985 dosed mice. At least seven metabolites were present in urine; one metabolite (constituting 8-14% of urinary radioactivity) was tentatively identified as the carboxylic acid resulting from the hydrolysis of the isopropyl group from the parent compound. In summary, NSC 615985 was poorly absorbed following oral administration and extensively metabolized and eliminated following i.v. or s.c. administration. This unfavorable pharmacokinetic profile of NSC 615985 as well as its pattern of activity against NNRTI-resistant strains of HIV-1 precluded its progression to clinical trial; however, other members of the general chemical class are currently being evaluated by the NCI.

Potent, selective, and orally bioavailable tricyclic pyridyl acetamide N-oxide inhibitors of farnesyl protein transferase with enhanced in vivo antitumor activity.

We previously reported compound 1 as a potent farnesyl protein transferase (FPT) inhibitor that exhibited reasonable pharmacokinetic stability and showed moderate in vivo activity against a variety of tumor cell lines. The analogous C-11 single compound, pyridylacetamide 2, was found to be more potent than 1 in FPT inhibition. Further studies showed that modification of the ethano bridge of the tricyclic ring system by conversion into a double bond with concomitant introduction of a single bond at C-11 piperidine resulted in compound 3 that had superior FPT activity and pharmacokinetic stability. Compound 4, a 5-bromo-substituted analogue of 3, showed improved FPT activity, had good cellular activity, and demonstrated a remarkably improved pharmacokinetic profile with AUC of 84.9 and t1/2 of 82 min. Compound4 inhibited the growth of solid tumor in DLD-1 model by 70% at 50 mpk and 52% at 10 mpk.

Inhibitors of farnesyl protein transferase. 4-Amido, 4-carbamoyl, and 4-carboxamido derivatives of 1-(8-chloro-6,11-dihydro-5H-benzo[5,6]- cyclohepta[1,2-b]pyridin-11-yl)piperazine and 1-(3-bromo-8-chloro-6,11- dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl)piperazine.

The synthesis of a variety of novel 4-amido, 4-carbamoyl and 4-carboxamido derivatives of 1-(8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl) piperazine to explore the SAR of this series of FPT inhibitors is described. This resulted in the synthesis of the 4- and 3-pyridylacetyl analogues 45a and 50a, respectively, both of which were orally active but were found to be rapidly metabolized in vivo. Identification of the principal metabolites led to the synthesis of a variety of new compounds that would be less readily metabolized, the most interesting of which were the 3- and 4-pyridylacetyl N-oxides 80a and 83a. Novel replacements for the pyridylacetyl moiety were also sought, and this resulted in the discovery of the 4-N-methyl and 4-N-carboxamidopiperidinylacetyl derivatives 135a and 160a, respectively. All of these derivatives exhibited greatly improved pharmacokinetics. The synthesis of the corresponding 3-bromo analogues resulted in the discovery of the 4-pyridylacetyl N-oxides 83b (+/-) and 85b [11S(-)] and the 4-carboxamidopiperidinylacetamido derivative 160b (+/-), all of which exhibited potent FPT inhibition in vitro. All three showed excellent oral bioavailability in vivo in nude mice and cynomolgus monkeys and exhibited excellent antitumor efficacy against a series of tumor cell lines when dosed orally in nude mice.

Metabolism and disposition of dimethyl hydrogen phosphite in rats and mice.

A study of dimethyl hydrogen phosphite (DMHP) by the National Toxicology Program (NTP) indicated that chronic administration by oral gavage resulted in an increased incidence of neoplastic lesions in the lungs and forestomachs of Fischer 344 rats but not in B6C3F1 mice. The current study was designed to evaluate the metabolic basis, if any, of this species selectivity by studying the metabolism and disposition of [14C]DMHP in the respective strains of rats and mice. Results of this study indicate that DMHP administered at a range of dose of 10-200 mg/kg was readily and near completely absorbed from the gastrointestinal tracts of rats and mice. DMHP-derived radioactivity was eliminated primarily as CO2 in the expired air, 44-57%, and urine, 28-49%, and very little was collected in feces, 1-2%, or as volatile organics, 2-3%. DMHP-derived radioactivity was widely distributed in tissues of rats and mice, with the highest concentrations observed in the liver, kidneys, spleen, lungs, and forestomach, and the lowest in brain, skeletal muscle, and adipose tissue. The disappearance of radioactivity from mouse tissues was approximately twice as rapid as from rat tissues. In vitro, DMHP was metabolized to formaldehyde by the microsomal fractions of liver, lungs, kidneys, forestomach, and glandular stomach. In vivo, DMHP was metabolized to the product of demethylation, monomethyl hydrogen phosphite (MMHP), which was excreted in urine. Results of this study indicate that the NTP carcinogenicity study with DMHP was carried out within the dose range in which the absorption, metabolism, and disposition of DMHP are linear in both species. Apparent species-dependent differences in the metabolism and disposition of DMHP are limited to the more rapid metabolism and elimination by the mouse. Therefore, the species-dependent variations in the carcinogenicity of DMHP are most likely attributable to factors other than metabolism and disposition.